Determining an analyte by multiple measurements through a cuvette

ABSTRACT

A method for measuring the presence or concentration of an analyte in a sample by spectrophotometry: providing an open top cuvette having a sample with an analyte to be measured; providing a light source and a detector for detecting emitted light; taking at least two measurements that includes: (i) directing at least two beams of light from the light source to different locations on the cuvette; (ii) passing the at least two beams through the cuvette at their respective locations and through the sample to be measured; and (iii) measuring at least two respective emitted light beams with the detector; and comparing the at least two emitted light beams to determine if: all the emitted light beams should be disregarded; one or more of the emitted light beams should be disregarded; or the sample absorbances should be averaged. In a preferred embodiment, the method includes taking at least three measurements. In another preferred embodiment, the spectrophotometry is absorption spectrophotometry, and the method is performed on a diagnostic analyzer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority as a continuation-in-part to Ser. No.10/784,505, filed Feb. 23, 2004, the contents of which are incorporatedherein by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates to measuring the presence or concentrationof an analyte in a sample, particularly by spectrophotometry on adiagnostic analyzer. In particular, the present invention relates toreducing the number of rejects or re-runs in measuring the concentrationof an analyte in a sample by taking multiple measurements through thecuvette containing the sample and reagent.

Known diagnostic assays and other analysis that use cuvettes as thereaction chamber or container for taking measurements often haveproblems with imprecise results associated with measurements of emittedlight, such as absorbance measurements, that are influenced byinterfering objects in the measurement path. These interfering objects,which can be transient or non-transient, can include any number ofthings from dirt or dust in the cuvette, dirt or dust on the exterior ofthe cuvette window, fingerprints on the surface of the cuvettes and airbubbles in the fluid. In addition to interfering objects, measurementerror and therefore imprecision of diagnostic assays performed in acuvette can be influenced by measuring a fluid, e.g., sample, that wasnot homogeneously mixed (chemically or thermally). The problems ofinterfering objects can be exacerbated by open top cuvettes which areopen to receiving fluids (e.g., sample and/or reagents) from adispensing or aspirating pipette or proboscis and are thus open to theintroduction of dirt from the ambient environment and additional bubblesfrom the dispense of fluid into the cuvette. The present inventors havefound that these transient conditions can be substantial contributors toassay imprecision which often leads to the assay being rejected, thusresulting in the time consuming and costly reanalysis (re-running) ofsamples. Some of these factors can be reduced by controlling theanalysis process. For example, mixing within the cuvette can be improvedas disclosed in pending application Ser. No. 10/622,258 filed Jul. 18,2003 entitled “Improved Fluid Mixing.” Cuvette loading can be improvedto reduce dirt and fingerprints as disclosed in pending application Ser.No. 10/684,536 filed Oct. 14, 2003 entitled “Packaging Of Multiple FluidReceptacles.”

A more difficult problem to eliminate or reduce is the formation of airbubbles in the fluid. The bubbles can be introduced by air being mixedin during sample or reagent dispense. Alternatively, air bubbles can beformed in the fluid because the fluid has more dissolved air presentwhen it is cold than when it is warm, and the reagents, which are storedcold, are warmed up in the cuvettes. As a result, bubbles of air tend toform on the surfaces of the cuvette as the reagents are warmed. If theyare located in the measurement window part of the cuvette they may causesubstantial error in the measurement and ultimately in the determinationof the assay concentration.

U.S. Pat. No. 4,123,173 discloses a rotatable flexible cuvette array.U.S. Pat. No. 4,648,712 discloses a method for determining the basisweight of a fibrous web that includes reading multiple areas of web.U.S. Pat. No. 4,549,809 discloses curved cuvettes and taking multiplereadings to determine the position of the cuvette and using a singlemeasurement for analysis. U.S. Pat. No. 5,402,240 discloses a spermdensimeter that takes a plurality of sample transmission measurementsand calculates an average based on the plurality of measurements. U.S.Pat. No. 5,535,744 discloses an analysis method that includes multiplereads for each cuvette which are averaged to determine a final result.U.S. Pat. No. 5,255,514 discloses a method for determining washeffectiveness on a dry slide test element that includes reading atdifferent locations on the slide. U.S. Pat. No. 5,853,666 discloses asealed test card having a plurality of wells containing sample to beanalyzed by fluorescence. Measurements are taken at multiple positionsacross the well to detect any air pockets or debris and to detect andreject abnormal transmittance measurements.

None of the known art described above, adequately addresses resolvingthe problems described above, in particular, of improving precision ofmeasurements through a cuvette to reduce or even eliminate the number ofre-runs that have to be performed on a sample, in particular, bydetecting and reducing or eliminating errors in reading through acuvette. For the foregoing reasons, there is a need for a method ofimproving precision, more particularly detecting and reducing oreliminating errors during measurement of an analyte byspectrophotometry.

SUMMARY OF THE INVENTION

The present invention is directed to a method that solves the foregoingproblems of improving precision, in particular in detecting andeliminating or reducing errors to reduce the number of samples that haveto be re-run and hence the time and cost of analysis. In someembodiments, the present invention also results in improvement in theaccuracy of results. One aspect of the invention is directed to a methodfor measuring the presence or concentration of an analyte in a sample byspectrophotometry, which includes: providing an open top cuvette havinga sample with an analyte to be measured; providing a light source and adetector for detecting emitted light; taking at least two measurementsthat includes: (i) directing at least two beams of light from the lightsource to different locations on the cuvette; (ii) passing the at leasttwo beams through the cuvette at their respective locations and throughthe sample to be measured; and (iii) measuring at least two respectiveemitted light beams with the detector; and comparing the at least twoemitted light beams to determine if: all the emitted light beams shouldbe disregarded; one or more of the emitted light beams should bedisregarded; or the sample absorbances should be averaged. In apreferred embodiment, the method includes taking at least threemeasurements and comparing the at least three emitted light beams todetermine if: all the emitted light beams should be disregarded; one ormore of the emitted light beams should be disregarded; or the emittedlight beams should be averaged. In another preferred embodiment, thespectrophotometry is absorption spectrophotometry.

In a preferred embodiment, prior to the step of directing at least twobeams, the method further includes: (i) directing at least two beams oflight from the light source at their respective different locations onthe cuvette; (ii) passing the at least two beams through the cuvettealone or the cuvette and sample before the sample has reacted withreagents; and (iii) measuring at least two respective blank absorbancesfrom the emitted light corresponding to the at least two beams with thedetector; and selecting at least one blank absorbance; and subtractingat least one blank absorbance from the at least two sample absorbancesto result in corrected sample absorbances. In a preferred embodiment,the analysis is performed on a diagnostic analyzer and the light has awavelength in the range of 300 to 1100 nm.

According to another aspect of the invention there has been provided amethod for measuring the presence or concentration of an analyte in asample by absorption spectrophotometry, which includes: providing acuvette having a sample with an analyte to be measured; providing asource of light and a detector for detecting the light; taking at leastthree measurements that includes: (i) directing at least three beams ofthe light to different locations on the cuvette; (ii) passing the atleast three beams through the cuvette at their respective locations andthrough the sample to be measured; and (iii) measuring at least threerespective sample absorbances of the transmitted beams with thedetector; and comparing the at least three sample absorbances todetermine if: all the sample absorbances should be disregarded; one ormore of the sample absorbances should be disregarded and the remainingabsorbances retained; or all the sample absorbances should be averaged,wherein: if at least two sample absorbances are retained and an averageretained absorbance is less than a first selected absorbance then thelowest absorbance is used in determining the presence or concentrationof the analyte; or if at least two sample absorbances are retained andan average retained absorbance is greater than or equal to a secondselected absorbance then the highest absorbance is used in determiningthe presence or concentration of the analyte.

According to yet another aspect of the invention, there has beenprovided a method for measuring the presence or concentration of ananalyte in a sample by absorption spectrophotometry. The methodincludes: (A) providing a cuvette having a sample with an analyte to bemeasured; (B) providing a source of light and a detector for detectingthe light; (C) taking at least three measurements that includes: (i)directing at least three beams of the light to different locations a, band c on the cuvette; (ii) passing the at least three beams through thecuvette at their respective locations a, b and c and through the sampleto be measured; and (iii) measuring at least three respective sampleabsorbances Aa, Ab and Ac of the transmitted beams with the detector;(D) determining the absolute value of the difference between each pairof absorbances to arrive at |Aa−Ab|, |Ac−Ab| and |Ac−Aa|; (E) comparingan absolute value of the difference between each pair of absorbanceswith a predetermined limit; (F) if one or more of each the absolutevalue of the difference is≧the predetermined limit, then compare eachabsorbance to a predetermined absorbance: (i) if one or more absorbancesare above the predetermined absorbance, then disregard all readings andproceed to step (K); or (ii) if all absorbances are below thepredetermined absorbance, then (G) determine the smallest absolute valueof the difference between each pair of absorbances; (H) determine if thesmallest absolute value of the difference is<a predetermined fraction ofthe predetermined limit: (i) if the smallest absolute value of thedifference is not less than the predetermined fraction of the limit thendisregard all readings and proceed to step (K); or (ii) if the smallestabsolute value of the difference is less than the predetermined fractionof the limit, then (I) determine which of the absolute value of thedifference between each pair of absorbances is the smallest absolutevalue of difference; (J) determine which absorbance in the smallestabsolute value should be selected or if the results should bedisregarded; and (K) either re-evaluating the analysis if the resultsshould be disregarded in steps (F), (H) or (J), or calculating thepresence concentration of the analyte in the sample by using theselected absorbance.

In a preferred embodiment, in the method described above, prior to thestep of directing at least three beams, the method further includes: (i)directing at least three beams of the light at their respectivedifferent locations a, b and c on the cuvette; (ii) passing the at leastthree beams through the cuvette alone or the cuvette and sample beforethe sample has reacted with reagents; (iii) measuring at least threerespective blank absorbances A1 a, A1 b and A1 c of the transmittedbeams with the detector; (iv) determining the sample absorbance Aa, Ab,and Ac by subtracting the blank absorbance A1 a, A1 b and A1 c frommeasured sample absorbance A2 a, A2 b and A2 c, respectively; whereinthe step of determining which read in the smallest absolute value of thedifference between each pair of absorbances should be selected or if theresults should be disregarded (J) comprises: (J11) if the smallestabsolute value of the difference between each pair of absorbances is|(A2 a−A1 a)−(A2 b−A1 b)|, then if A1 c+A2 c is greater than each of A1a+A2 a and A1 b+A2 b, compare A1 a+A2 a and A1 b+A2 b, if A1 a+A2 a<A1b+A2 b then absorbance Aa is the selected absorbance, otherwiseabsorbance Ab is the selected absorbance, if A1 c+A2 c is≦to one of A1a+A2 a and A1 b+A2 b, then disregard all readings and proceed to step(K); (J2) if the smallest absolute value of the difference between eachpair of absorbances is |(A2 c−A1 c)−(A2 b−A2 b)|, then if A1 a+A2 a isgreater than each of A1 b+A2 b and A1 c+A2 c, compare A1 c+A2 c and A1b+A2 b, if A1 c+A2 c<A1 b+A2 b then absorbance Ac is the selectedabsorbance, otherwise absorbance Ab is the selected absorbance, if A1a+A2 a is≦to one of A1 b+A2 b and A1 c+A2 c, then disregard all readingsand proceed to step (K); or (J3) if the smallest absolute value of thedifference between each pair of absorbances is |(A2 c−A1 c)−(A2 a−A1a)|, then if A1 b+A2 b is greater than each of A1 a+A2 a and A1 c+A2 c,compare A1 c+A2 c and A1 a+A2 a, if A1 c+A2 c<A1 a+A2 a then absorbanceAc is the selected absorbance, otherwise absorbance Aa is the selectedabsorbance, if A1 b+A2 b is≦to one of A1 a+A2 a and A1 c+A2 c, thendisregard all readings and proceed to step (K).

According to another aspect of the invention, the method described aboveis implemented by a computer program interfacing with a computer.Another aspect of the invention provides an article of manufacturecomprising a computer usable medium having computer readable programcode configured to conduct the method described above.

Further objects, features and advantages of the present invention willbe apparent to those skilled in the art from detailed consideration ofthe preferred embodiments that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing one embodiment of the measurementwindow of a cuvette with three measurements at different locations onthe window.

FIG. 2 is a flow diagram showing the logic of an algorithm fordetermining whether one or more absorbances should be retained or theanalysis re-evaluated according to a preferred embodiment of the presentinvention.

FIG. 3 is a graphical representation of patterns of absorbances or readsthat would pass the criteria of Decision Block 5 of FIG. 2.

FIG. 4 is a graphical representation of patterns of absorbances or readsthat would fail the criteria of Decision Block 5 of FIG. 2.

FIG. 5 is a graphical representation of patterns of absorbances or readsthat would pass the criteria of Decision Block 8 of FIG. 2.

FIG. 6 is a graphical representation of patterns of absorbances or readsthat would fail the criteria of Decision Block 8 of FIG. 2.

FIG. 7 is a graphical representation of patterns of absorbances or readsthat would pass the criteria of Decision Block 10 of FIG. 2.

FIG. 8 is a graphical representation of patterns of absorbances or readsthat would fail the criteria of Decision Block 10 of FIG. 2.

FIG. 9 is a graph showing the measurement of the concentration ofC-reactive protein in 36 cuvettes with 3 measurements for each cuvette.

FIG. 10 is a graph showing the standard deviation of absorbance at threedifferent locations on the cuvettes and the minimum absorbance on eachcuvette using three different threshold discards for the measurementsshown in FIG. 9.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention includes a method for measuring an analyte in asample by spectrophotometry, including a method for detecting one ormore errors during the measurement of a sample and then applying anappropriate correction if an error is detected. Broadly, the methodinvolves providing a light source which directs a beam of light from thelight source (defined below) through the sample to be measured at leasttwo different locations in the cuvette, containing the sample, andmeasuring the amount of light emitted from the cuvette and sample. Themeasurements are compared with one another. Based upon the comparison,in particular the difference in the measurements of emitted light ofthese samples, one can determine whether there has been: an error in oneor more of the measurements and take appropriate action, such asdiscarding or disregarding one or more of the measurements as an outlierand using the remaining measurements for the analysis, or alternativelydisregarding all measurements and either remeasuring the sample in thesame cuvette or preparing a new sample for measurement; or whether thereare no significant errors such that all measurements are consideredacceptable, in which case, all measurements can be used, or morepreferably one of the measurements can be used, e.g., the highest orlowest, depending on the type of analysis being conducted.

The present invention thus solves the problems of optically interferingconditions affecting the measurement of a sample through a cuvette byboth detecting interfering conditions and determining how the data fromall of the measurements should be treated in order to reduce the numberof sample re-runs that have to be performed. As used herein an“interfering condition(s)” is anything other than a uniform error in thecuvette (i.e. path-length error) or the sample that will cause anincrease or decrease in the emitted light in the measurement area of thecuvette, which condition does not extend across the entire read area ofthe cuvette. Interfering condition(s) can include permanent interferentssuch as a spatial defect in the cuvette and fingerprints on the surfaceof the cuvettes, or transient interferents, such as dirt or dust in thecuvette, dirt or dust on the exterior of the cuvette window, air bubblesin the fluid or sample that was not homogeneously mixed (chemically orthermally).

A significant advantage of the present invention is that precision ofanalysis can be improved without necessarily eliminating the factorscontributing to the imprecision, such as bubbles, etc. Even moresignificantly, an advantage of the present invention is that a greaternumber of sample analysis will be useable in spite of the fact thatfactors leading to outlying reads may be present (i.e., there will beless of a requirement for re-running sample and the time and expenseassociated therewith). The present invention also allows the user todetermine if the uncertainty of the quality of the results is highenough such that the results should be disregarded, thus requiring theuser to re-run the analysis using a new sample aliquot.

The method of the present invention can be used in any analysismethodology and analyzer that includes detecting light from a sample tobe measured and is broadly referred to herein as spectrophotometry. Someexamples include absorption spectrophotometry assays such as end-pointreaction analysis and rate of reaction analysis, turbidimetric assays,nephelometric assays, radiative energy attenuation assays (such as thosedescribed in U.S. Pat. Nos. 4,496,293 and 4,743,561 and incorporatedherein by reference), ion capture assays, colorimetric assays, andfluorometry spectrophotometry assays, and immunoassays, all of which arewell known in the art. A preferred analysis technique is absorptionspectrophotometry such as end-point reaction analysis and rate ofreaction analysis. The preferred embodiments of the present inventionare described with reference to absorption spectrophotometry althoughthe broad aspect of the present invention is not so limited.

The sample generally contains an analyte being measured, preferably in adiagnostic assay. Examples include, HDL (high density lipoprotein),which is a generally a two point rate assay. Another example is highsensitivity CRP (C-reactive protein), which is generally a blankedendpoint assay. Still another example is Gentamicin, which can generallybe done as an endpoint assay, two point rate assay or multipoint rateassay. However, other analytes can also be measured, such as a chemicalanalyte in an organic or inorganic medium in an industrial setting, forexample, in a quality assurance laboratory or an environmental analysis.

A cuvette is provided for containing the sample. In a preferredembodiment, the cuvette is an open top cuvette adapted for receiving thetip of a pipette or proboscis which dispenses or aspirates sample and/orreagents into the cuvette, such as those described for example in U.S.Patent Application Publication No. 2003/0003591 A1, Des. 290,170 andU.S. Pat. No. 4,639,135, all of which are incorporated by reference intheir entireties. Particularly preferred are cuvettes having a pluralityof vertically disposed reaction chambers side-by-side in spacedrelation, each of said reaction chambers having an open top and beingsized for retaining a volume of sample or reagent as described in the'591 published application.

A source of light and a detector are also provided. The wavelength oflight used preferably ranges from mid infrared (approx. 1100 nm) toultraviolet (approx. 300 nm) depending on the analysis to be performed.The light source can be any well known source such as a photodiode. Thedetector can be detectors well known for the particular method ofanalysis. For example, in a spectrophotometric method, the detector canbe a photodiode or a charged couple device (CCD), such as a 2 to 5 megapixel detector.

As noted above, at least two measurements are taken through the sampleand cuvette at different spatial locations. The number of measurementscan range from 2 up to millions in the case of a mega pixel CCD. Theonly limitation on the number of measurements is the physical limitationon placing the light source(s) and detector(s) in a proper position withthe sample and cuvette to be measured. In a preferred embodiment 3 to 5measurements are taken through the sample, with 3 measurements being themost preferred, e.g., at locations a, b and c on the cuvette, whichpreferably correspond to Left (L), Right (R) and Middle (M) positions.It is important that the measurements be taken at different spatiallocations to avoid measuring the same interfering condition(s) (e.g. anair bubble) at all the same measurement locations. To achievemeasurements at different locations across the cuvette, a single lightsource is preferably held stationary, while the cuvette is movedrelative to the light source. Of course, a single light source may bemovable, or multiple stationary lights source may be employed.

As shown in FIG. 1, in a preferred embodiment, multiple measurements aretaken spatially across the cuvette (10). As noted above, these spatialmeasurements are intended to both determine if there is reason todiscard the result from this cuvette or to merge the data in a way toproduce a more consistent result. In the embodiment shown in FIG. 1, themeasurements are spaced 0.024″ apart for a total distance of 0.048″(across the three measurements) with a measurement window of 0.059″.This enables the detection of interfering condition(s) that are uniqueto particular areas of the cuvette.

The light beams are transmitted through the cuvette and are partiallyabsorbed depending on the concentration of the analyte in the sample andother factors such as scattering and absorbance due to the interferingcondition. The transmitted portion of the beams are measured by thedetector which in the case of absorption spectrophotometry is generallylocated opposite where the beam of light enters the sample and cuvetteto result in a sample absorbance A.

An important aspect of the invention is that instead of simplycalculating an average sample absorbance based on the multiplemeasurements, as is done in the known art, the measurements or sampleabsorbances are compared with one another to determine if at least oneof the measurements has been affected by an interfering condition(s) orcontaminate. Based on the comparison of sample absorbances, the samplemay be handled in the following manner depending on the analysis beingcarried out: (i) all the sample absorbances may be averaged; (ii) atleast one of the sample absorbances may be disregarded and at least oneof the other sample absorbances used alone or averaged with anotheracceptable sample absorbance; and (iii) all the sample absorbancesshould be disregarded, with the particular sample aliquot of samplebeing re-measured or discarded and a new sample aliquot being re-run.

In a preferred embodiment, the comparison of sample absorbances iscarried out by determining the difference between the comparedabsorbances. This difference is then compared to a selected absorbancedifference. If the absorbance differences between any one of themeasurements exceeds the selected absorbance difference, further actionis then undertaken as described above and further described below inconnection with the preferred embodiments.

In some embodiments, including both endpoint and rate assays describedbelow, a blank measurement A1 may be taken before the sample measurementA2. That is, a blank measurement may be taken before any sample is addedto the cuvette, or before reagent is added to the sample already in thecuvette. In the case of slow reactions, it may be possible to measurethe blank absorbance after reagents have been added to the sample, butbefore any significant reaction has taken place. After the blankmeasurement is obtained, the sample measurement is carried out afteradding sample and/or reagent and providing sufficient time for mixingand reaction. The blank absorbance is subtracted from the sampleabsorbance to yield a corrected absorbance A. The blank measurement willgenerally contribute to a reduction in some errors, by canceling outerrors that are continually present during the analysis, such as markson the cuvette (e.g., fingerprint smudges) or defects in the cuvette.For example, if a mark on the cuvette in the area of one of the spatialmeasurements contributes to a 0.03 increase in absorbance, this increasewill be present during both the blank and sample measurement.Subtracting the blank absorbance from the sample absorbance will thencancel the 0.03 increase. If no blank measurement is carried out, thenthe method of the present invention would flag the one measurement as anoutlier and take further action as appropriate (e.g., discard theoutlier or the entire measurement for that sample). The blankmeasurement embodiment may be used with the other embodiments of thepresent invention.

The present invention can be used in both endpoint or rate assays. Bothof these assays are well known in the field of spectrophotometry. See,e.g., Modern Optical Methods of Analysis by Eugene D Elson 1975, whichis incorporated by reference in its entirety. Briefly stated, anendpoint assay takes a single measurement (not including a blankmeasurement) after reaction between sample and reagents. That is, afterdevelopment of the chromophore that will absorb the light beingtransmitted through the sample. Using the present invention with theendpoint assay technique simply requires that the sample measurements,at the different spatial locations on the cuvette, be taken only once,generally after complete development of the chromophore. Thesemeasurements are compared with one another according to the presentinvention.

On the other hand, a rate assay will take at least two measurements foreach spatial location at different times after the reagent has beenadded. Rate assays provide much more data and flexibility. Testing hasshown that deliberate interfering condition(s) such as defects or marksmade on the surface of the cuvette produce no impact on calculated rateeven when these differences are large for the same reason that a blankmeasurement will result in a reduction of errors. That is, in bothassays that include blank measurements and rate assays, a difference inabsorbance is being measured, which will cancel out increased absorbance(or decreased absorbance in the case of high absorbances) due to theinterfering condition(s), unless the interfering condition obstructslight to the point that the spectrophotometer noise becomes an issue.Based on the different measurements at different times, a rate for eachspatial measurement location on the cuvette can be determined. Todetermine errors, the difference in rates are compared for the variousmeasurement locations.

As noted above, an important feature of the present invention lies in acomparison of the measurements at each different spatial location acrossthe cuvette to determine or detect if an error exists. Based on thecomparisons, many different courses of action are available as describedin connection with preferred embodiments below.

In one embodiment, each sample absorbance is compared with the othersample absorbance(s). If the difference between any of the absorbancesexceeds a selected difference in absorbance, all of the measurements arediscarded and the same sample/cuvette is remeasured. Alternatively, anew aliquot of sample or a new cuvette is used and measured. This isless preferred than other embodiments, since it likely entails thenecessity of re-running a new sample aliquot at additional time andexpense.

The selected difference in absorbance can be pre-determined based on theparticular analysis being carried out and the requirements for precisionand sensitivity. For example, in an assay that has a calibration curvewith a steep slope (i.e., a strong signal to noise ratio), a smallvariation in absorbance will result in a small change in the predictedconcentration of the analyte being assayed. Thus, less precision wouldbe required. In contrast, in an assay that has a calibration curve witha shallow slope (i.e., a weak signal to noise ratio), a small variationin absorbance will result in a significant change in predictedconcentration. Thus, more precision will be required and only arelatively small difference in absorbances is generally acceptable.

Alternatively, the selected difference may be determined by the CPUcontrolling the analyzer during the measurements of the sample(s). Suchselection by the CPU may be based on specifications inputted by theoperator or software controlling the CPU, and/or trends observed by theCPU during the measurements of multiple samples. For example, the CPUmay determine that a greater degree of imprecision will be tolerated fora certain assays, based on previous knowledge of the assay calibrationcurve slope. That is, as described above, an assay with a large signal(i.e., a steep calibration curve) will tolerate a greater degree ofimprecision and thus the selected difference may be greater, while thoseassays with less signal (i.e., a shallow calibration curve) will requiregreater precision and thus the selected difference will be less.

If the difference in absorbances is within the selected difference, thenall of the absorbance measurements can be averaged and the averageabsorbance is used in the calculation of the concentration of thesubstance to be measured in the sample. Alternatively, as describedbelow, one of the absorbances (generally the highest or lowestabsorbance) can preferably be selected to determine concentration. Inthis embodiment, the analysis can be carried out with or without a blankmeasurement as described above.

In another preferred embodiment, each sample absorbance is againcompared with the other sample absorbance(s), preferably all of theother sample absorbances, to determine a difference in absorbances. Ifthe difference between all of the absorbances exceeds a selecteddifference in absorbance, all of the measurements are discarded and thesame sample/cuvette is remeasured. Alternatively, a new aliquot ofsample or a new cuvette is measured.

If at least two of the absorbances have differences which are less thanthe selected difference, these absorbances are used in the calculationof the concentration of the substance being measured. As noted in theembodiment described above, these absorbance measurements can beaveraged and the average absorbance is used in the calculation of theconcentration of the substance to be measured in the sample.Alternatively, as described below, one of the absorbances (generally thehighest or lowest absorbance) can be selected to determineconcentration. In this embodiment, the analysis can be carried out withor without a blank measurement as described above.

The selected difference in absorbance can be determined eitherbeforehand or during the analysis by the CPU as described above. Thisembodiment can be used with or without a blank measurement.

In another preferred embodiment, blank measurements are taken at leasttwo different spatial locations across the cuvette, preferably at thesame locations that one or more of the sample measurements will becarried out. The blank absorbances obtained by the blank measurementsare then compared with a selected threshold blank absorbance. If theblank absorbance measurements are below the selected threshold blankabsorbance value, then one or all of the blank measurements can be usedin the further analysis. For example, each blank measurement can besubtracted from its corresponding sample measurement. Alternatively, thelowest blank absorbance or an average blank absorbance can be subtractedfrom all sample measurements. If one or more blank absorbances,particularly one blank absorbance, are above the threshold absorbance,then this is evidence that a bubble or other interfering condition(s) ispresent and these blank absorbances should be discarded. The selectedthreshold absorbance can be predetermined based on previous experiencewith a particular sample or substance being measured. Alternatively, theselected threshold absorbance can be determined by the analyzer whilethe samples are being run based on state of the samples, previousanalysis of samples, etc.

In a particularly advantageous aspect of the invention, the inventorshave discovered that in low absorbance (e.g., an absorbance<one (1)absorbance unit) assay embodiments, the most accurate result generallyoccurs when the lowest of the at least three sample absorbances(optionally corrected with a blank measurement) for a rate or end pointcalculation is used. This lowest absorbance measurement is preferablyonly selected if, for endpoint assays, the three measurements are withinan acceptable threshold, or for rate assays, the calculated ratedifference from measurement position to measurement position is withinan acceptable threshold using the techniques described above. That is,in the same manner above, the at least three sample absorbances arecompared to determine if: all the sample absorbances should bedisregarded; one or more of the sample absorbances should be disregardedand the remaining absorbances retained; or all the sample absorbancesshould be averaged. If at least two sample absorbances are retained andan average retained absorbance is less than a selected absorbance thenthe lowest absorbance is used in determining the presence orconcentration of the analyte.

While not being bound by any theory, the inventors believe that thereason for selecting the lowest absorbance measurement for relativelylow absorbance assays is that interfering condition(s) have been shownto only increase absorbance. That is, the absorbance caused by theinterfering condition(s) relative to the relatively low absorbance ofthe sample is higher. Thus, the higher absorbance measurements are morelikely to be erroneous, since these are more likely due to theinterfering condition(s), and the more accurate result will be obtainedusing the lower absorbance measurements. Conversely, at some thresholdof absorbance, which can be determined by those skilled in the artthrough routine experimentation, interfering condition(s) will tend todecrease the measured absorbance. As a result, at high absorbancemeasurements, the higher absorbance measurements are more likely to berepresentative of the true concentration, since the lower absorbancemeasurements are more likely due to the transient defect. That is, inthe same manner above, the at least three sample absorbances arecompared. If at least two sample absorbances are retained and an averageretained absorbance is greater than or equal to a selected absorbancethen the highest absorbance is used in determining the presence orconcentration of the analyte.

In a preferred embodiment, the threshold or cutoff absorbance is one (1)absorbance unit (AU). That is for absorbances that are less than one,the lowest absorbance is used in the determination, whereas forabsorbances that are greater than or equal to one (1) absorbance unit(AU), the highest absorbance is used in the determination.

As described above, in certain embodiments, if at least two absorbanceshave a difference in absorbance which is less than a selecteddifference, then one or an average of the at least two absorbances canbe used in the calculation of the concentration of the analyte orsubstance being measured. In the embodiment described below, the presentinvention provides a method that can determine which, if any,absorbances can be used if the at least two absorbances have differencewhich is less than a predetermined limit, which is dependent on the testor assay being performed. The method of this preferred embodimentemploys an algorithm that can be used in applications where any one ofthe three absorbances (in those analysis where three absorbances areemployed) is sufficiently different from the other two (i.e., outsidethe selected difference in absorbance), and which scrutinizes theresults if there are two absorbances that agreed (i.e., are within theselected difference in absorbance) to determine if the result isreportable or might be “salvaged,” (i.e., the analysis does not have tobe re-run).

The algorithm used in the method of this embodiment (“hereinafterreferred to as “Algorithm”) examines absorbances for certain patterns todetermine if the differing or outlying absorbance was really the oneaffected by a possible interfering condition, such as a bubble or debrisin the cuvette cell. If certain pattern conditions are met, a predictioncan be made from the two absorbances in agreement. The importantcondition that is addressed by the Algorithm is one where twoabsorbances agree, but in fact are both affected by an interferingcondition, e.g., bubbles or debris, in a manner that affects bothequally (i.e., the disagreeing absorbance or outlier is actually thecorrect absorbance). The pattern analysis logic of the Algorithmidentifies such events. Data suggests that this is a rare event, but onethat is important to detect and report as a “no result” should it beencountered, requiring a re-evaluation of the analysis that can includere-running the same sample or analyzing a new aliquot of sample, or someother mode of intervention by the operator.

FIG. 2 is a block diagram detailing the logic of the Algorithm.Definitions are provided as follows (in the Figures and below, “read”and “absorbance” are used interchangeably):

Axy=Read identifier, x=1 (first read, e.g., blank), 2 (second read);y=a, b, c (read position, preferably L, M, R, left, middle, or right).Example: “A1M”=Read 1, middle position. “A2 c”=Read 2, position c.Left Response (Resp 1)=A2L-A1L. If the math model is a rate assay, thenLeft Response (Resp 1)=(A2L-A1L)/(Read 2 time-Read 1 time).MidResponse (Resp 2)=A2M-A1M. If the math model is a rate assay, thenMid Response (Resp 2)=(A2M-A1M)/(Read 2 time-Read 1 time).RightResponse (Resp 3)=A2R-A1R. If the math model is a rate assay, thenRightResponse (Resp 3)=(A2R-A1R)/(Read 2 time-Read 1 time).Limit (corresponds to the predetermined limit describedabove)=intercept+slope*(minimum of Resp1, Resp2, or Resp3); note thatintercept and slope are assay specific.Delta 1=Resp1−Resp2. Alternatively, “Delta” can be described as thedifference between each pair of absorbances.

Delta2 =Resp2−Resp3. Delta3=Resp3−Resp1.

Min Delta=min(abs(Delta1), abs(Delta2), abs(Delta3)). Alternatively, theabsolute minimum of Delta can be represented by e.g., |Delta1| or|(A2L−A1L)−(A2M−A1M)| using standard mathematical symbols.Max Delta=max(abs(Delta1), abs(Delta2), abs(Delta3))Fail Response=a condition where the three absorbances do not meetacceptance checks of the Algorithm. In this event, “no result” isreported for the test rather than a concentration prediction and theanalysis must be re-evaluated.

The numbered circular bubbles in the FIG. 2 Algorithm Flow Chart (e.g.{circle around (1)}) correspond to the following description in points 1through 11 below. Although the description below is with reference toleft, right and middle positions (L, R, M), the present invention is notso limited. For example, the position could be top, center and bottom.Thus, an alternative designation of description can be location a, b andc as described above.

1. The first Decision Block indicated by 1 is a check to see if Delta1,Delta 2, and Delta3 are all less than the Limit. If so, the result ispredicted from the center absorbance in a preferred embodiment. If thiscondition is not satisfied, the Algorithm moves to decision point 2 forfurther evaluation of the data (and possibly “salvaging” the result).Prior to the present invention, the analyzer would have simply failedthe response and issued a “no result” resulting in the necessity tore-evaluate.

2. Decision Block 2 checks to see if all optical absorbances are lessthan 1.0 AU (optical absorbance units) for this particular embodiment.As noted above, in the lower range of AU levels (1.0 is a conservativecutoff), bubbles and particulates in the optical pathway of the cuvettetend to raise the AU level of the absorbance by scattering, backreflecting, or diffracting the incident light. This is an importantcheck as a precursor to the data pattern checks that are described inthis embodiment. If all absorbances do fall below 1.0 AU, then theevaluation process continues. If not, then the response is failed and a“no result” issued.

3. Decision Block 3 is a check to see whether or not the two absorbancesthat agree actually agree exceptionally well (within 75% of the Limitaccording to this embodiment). If so, then the then the evaluationprocess continues. If not, then the response is failed and a “no result”issued.

4. Decision Block 4 is the first of several checks to determine whichpair of the three absorbances is the one having the exceptionalagreement. If Delta1 corresponds to the MinDelta (i.e., the smallestabsolute value of the difference between each pair of absorbances), thenpatterns checks are initiated to determine if either LeftResponse orMidResponse might be used for a prediction. If Delta1 does notcorrespond to MinDelta, then decision block 7 performs a similar checkon Delta2.

5. Decision Block 5 is a pattern check performed to determine if eitherLeftResponse or MidResponse might be used for a prediction. Themathematics in this block determine if the sum of the right absorbancesare a.) greater than the sum of the middle absorbances and are b.)greater than the sum of the left absorbances. If this assessment istrue, then a prediction of the correct pair using either the left ormiddle absorbances will be performed (Decision Block 6). FIG. 3 showsexamples of patterns that would pass the Decision Block 5 criteria(concludes that A1R and/or A2R is elevated and may have been affected byan interfering conditions such as a bubble or debris in the cuvette). InFIGS. 3-8, the solid circles and squares represent the first absorbancesand the outlined circles and squares represent the second absorbances.The circles are absorbances unaffected by interfering conditions, e.g.,bubbles, debris, and the squares represent absorbances affected byinterfering conditions. FIG. 4 shows examples of patterns that wouldfail the Decision Block 5 criteria (concludes that the left and middleabsorbances are actually the absorbances that have been affected bybubbles or debris in the cuvette and happen to agree with one another bychance). A failure in this Decision Block results in a “failed response”(i.e., no result).

6. Decision Block 6 performs the final selection of the absorbance set(either left or middle in this embodiment) to serve as the response pairand ultimately make a result prediction with. The Decision Block selectsthe pair of absorbances having the lowest numerical sum, the idea beingthat this is the “cleanest” absorbance set (i.e., interfering conditionssuch as bubbles and debris only act to raise AU levels in the rangesdescribed in this embodiment).

7. Decision Block 7 is analogous to Decision Block 4. It checks to seeif Delta2 is the MinDelta. If so, then pattern tests are performed inDecision Block 8 that examines the nature of the left absorbance (sincethe middle and right absorbances agree the best). If not, then thealgorithm moves to Decision Block 10 that examines the nature of themiddle absorbance (since the left and right absorbances agree the best).

8. Decision Block 8 is analogous to Decision Block 5. It is a patterncheck performed to determine if either MidResponse or RightResponsemight be used for a prediction. The mathematics in this block determineif the sum of the left absorbances are a.) greater than the sum of themiddle absorbances and are b.) greater than the sum of the rightabsorbances. If this assessment is true, then a prediction using eitherthe middle or right absorbances will be performed (Decision Block 9).FIG. 5 shows examples of patterns that would pass the Decision Block 8criteria (concludes that A1L and/or A2L is elevated and may have beenaffected by a bubble or debris in the cuvette. FIG. 6 shows examples ofpatterns that would fail the Decision Block 8 criteria (concludes thatthe middle and right absorbances are actually the absorbances that havebeen affected by bubbles or debris in the cuvette and happen to agreewith one another by chance). A failure in this Decision Block results ina “failed response” (i.e., no result).

9. Decision Block 9 is analogous to Decision Block 6. It performs thefinal selection of the absorbance set (either middle or right) to serveas the response pair and ultimate make a result prediction with. TheDecision Block selects the pair of absorbances having the lowestnumerical sum, the idea being that this is the “cleanest” absorbance set(i.e., bubbles and debris only act to raise AU levels in the ranges ofthe present embodiment).

10. Decision Block 10 is analogous to Decision Blocks 5 and 8. It is apattern check performed to determine if either LeftResponse orRightResponse can be used for a prediction. The mathematics in thisblock determine if the sum of the middle absorbances are a.) greaterthan the sum of the left absorbances and are b.) greater than the sum ofthe right absorbances. If this assessment is true, then a predictionusing either the left or right absorbances will be performed (DecisionBlock 11). FIG. 7 shows examples of patterns that would pass theDecision Block 10 criteria (concludes that A1M and/or A2M is elevatedand may have been affected by a bubble or debris in the cuvette. FIG. 8shows examples of patterns that would fail the Decision Block 10criteria (concludes that the left and right absorbances are actually theabsorbances that have been affect by bubbles or debris in the cuvetteand happen to agree with one another by chance). A failure in thisDecision Block results in a “failed response” (i.e., no result) and theanalysis will have to be re-evaluated.

11. Decision Block 11 is analogous to Decision Block 6 and 9. Itperforms the final selection of the absorbance set (either left orright) to serve as the response pair and ultimate make a resultprediction with. The Decision Block selects the pair of absorbanceshaving the lowest numerical sum, the idea being that this is the“cleanest” absorbance set (i.e., bubbles and debris only act to raise AUlevels in the ranges of the present embodiment).

The Algorithm according to this preferred embodiment can reduce thenumber of tests flagged for a multiple, e.g., triple absorbance failure(no results) by as much as 50% over previous algorithms, such as (ifDeltaX (X=1, 2, 3)>Limit, then no result). In experiments done by theinventors there are no analysis where a result was saved that shouldhave been rejected.

EXAMPLE

An assay for C-reactive protein (CRP) having a known concentration of0.582 mg/dl was prepared and analyzed in 36 different cuvettes. For eachcuvette an absorbance measurement was taken in the left, center andright of the cuvette. The results for each measurement in each cuvetteis plotted in FIG. 9 with lines marked with diamonds (⋄) for the left,squares (▪) for the center and triangles (▴) for the right. As FIG. 9shows, there were significant outliers for cuvettes Nos. 1, 6 and 28 asshown on the x-axis. These were likely due to the presence of airbubbles in the cuvettes. Even though there were significant outliers inthese cuvettes, only the results in cuvette 1 would be rejected in aclinical setting, since a comparison between the absorbances would yielda difference that was outside an acceptable threshold. In the remainingresults with outliers, while the differences in absorbance between theright and other reads were significant, the difference in absorbancebetween the center and left read was within acceptable threshold. Thus,these results can be used in a clinical setting without the need tore-run the samples again. In addition, to improve the accuracy of theresults, the lowest absorbance measurement can be used to determine theconcentration of CRP, because of the low absorbance (<1) measurementsfor these samples. The higher absorbance readings (even those within anacceptable threshold of absorbance difference) were likely due to thepresence of interfering conditions.

FIG. 10 illustrates the example of FIG. 9 slightly differently.Specifically, FIG. 10 shows the standard deviation (SD) for differentlocations on the cell and for the minimum absorbance on each cell,regardless of read location. The line marked with diamonds (⋄) was thestandard deviation when all of the cells were included, including thesignificant outliers for cuvettes Nos. 1, 6 and 28 as shown in FIG. 9.As shown in FIG. 10, the standard deviation for all locations (left,center and right) and the minimum (for each cuvette) was greatest whenthe absorbance for all cuvettes were included. The line marked withtriangles (▴) was the standard deviation when only cuvette 1 wasexcluded from the standard deviation calculation. As shown in FIG. 10,the standard deviation for all locations and the minimum was less thanthe standard deviation that included all cuvettes. The line marked withsquares (▪) was the standard deviation when cuvettes 1, 6 and 28 wereexcluded. As shown in FIG. 10, the standard deviation for all locationsand the minimum was the least when cuvettes 1, 6 and 28 were excluded.

The measurement method according to the present invention can beimplemented by a computer program, having computer readable programcode, interfacing with the computer controller of the analyzer as isknown in the art.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the compounds, compositionsand processes of this invention. Thus, it is intended that the presentinvention cover such modifications and variations, provided they comewithin the scope of the appended claims and their equivalents.

The disclosure of all publications cited above are expresslyincorporated herein by reference in their entireties to the same extentas if each were incorporated by reference individually.

1. A method for measuring the presence or concentration of an analyte ina sample by spectrophotometry, comprising: providing an open top cuvettehaving a sample with an analyte to be measured; providing a light sourceand a detector for detecting emitted light; taking at least twomeasurements that includes: (i) directing at least two beams of lightfrom the light source to different locations on the cuvette; (ii)passing the at least two beams through the cuvette at their respectivelocations and through the sample to be measured; and (iii) measuring atleast two respective emitted light beams with the detector; andcomparing the at least two emitted light beams to determine if: all theemitted light beams should be disregarded; one or more of the emittedlight beams should be disregarded; or the emitted light beams should beaveraged.
 2. A method for measuring as claimed in claim 1, furthercomprising taking at least three measurements and comparing the at leastthree emitted light beams to determine if: all the emitted light beamsshould be disregarded; one or more of the emitted light beams should bedisregarded; or the emitted light beams should be averaged.
 3. A methodfor measuring as claimed in claim 1, wherein the spectrophotometry isfluorescence spectrophotometry.
 4. A method for measuring as claimed inclaim 1, wherein the spectrophotometry is absorption spectrophotometryand the step of taking at least two measurements includes: (i) directingat least two beams from the light source to different locations on thecuvette; (ii) passing the at least two beams through the cuvette attheir respective locations and through the sample to be measured; and(iii) measuring at least two respective sample absorbances from theemitted light corresponding to the at least two beams with the detector;and comparing the at least two sample absorbances to determine if: allthe sample absorbances should be disregarded; one or more of the sampleabsorbances should be disregarded; or the sample absorbances should beaveraged.
 5. A method for measuring as claimed in claim 1, wherein asingle light source and a single detector are provided and the cuvetteis moved relative to the light source and cuvette to produce the atleast two beams of light.
 6. A method for measuring as claimed in claim4, wherein prior to the step of directing at least two beams, the methodfurther comprises: (i) directing at least two beams of light from thelight source at their respective different locations on the cuvette;(ii) passing the at least two beams through the cuvette alone or thecuvette and sample before the sample has reacted with reagents; and(iii) measuring at least two respective blank absorbances from theemitted light corresponding to the at least two beams with the detector;and selecting at least one blank absorbance; and subtracting at leastone blank absorbance from the at least two sample absorbances to resultin corrected sample absorbances.
 7. A method for measuring as claimed inclaim 6, wherein a single light source and a single detector areprovided and the cuvette is moved relative to the light source andcuvette to produce the at least two beams of light.
 8. A method formeasuring as claimed in claim 6, wherein all blank absorbances areselected and each blank absorbance is subtracted from its correspondingsample absorbance at the same location.
 9. A method for measuring asclaimed in claim 6, wherein the lowest blank absorbance is selected andthe lowest blank absorbance is subtracted from each sample absorbance.10. A method for measuring as claimed in claim 4, wherein after a periodof time after the at least two measurements, the method furthercomprises: taking at least two second measurements at the same locationas the at least two measurements to result in at least two second sampleabsorbances; subtracting the at least two sample absorbances from thesecond sample absorbances to result in a rate sample absorbance.
 11. Amethod for measuring as claimed in claim 4, wherein the comparisonincludes comparing the sample absorbances with each other, and if adifference in absorbance between any two absorbances exceeds apredetermined absorbance, then disregarding all sample absorbances. 12.A method for measuring as claimed in claim 4, wherein the comparisonincludes comparing the sample absorbances with each other: if adifference in absorbance between all absorbances exceeds a predeterminedabsorbance, then disregarding all sample absorbances; if the differencebetween a predetermined number of absorbances, which is less than thetotal number of absorbances, is within the predetermined absorbance,then discarding the remaining absorbances and averaging the absorbancesof the predetermined number of absorbances.
 13. A method for measuringas claimed in claim 6, wherein the comparison includes comparing thecorrected sample absorbances with each other. if a difference inabsorbance between all absorbances exceeds a predetermined absorbance,then disregarding all sample absorbances; if the difference between apredetermined number of absorbances, which is less than the total numberof absorbances, is within the predetermined absorbance, then discardingthe remaining absorbances and averaging the absorbances of thepredetermined number of absorbances.
 14. A method for measuring asclaimed in claim 6, wherein the comparison includes comparing thecorrected sample absorbances with each other, and if a difference inabsorbance between any two corrected sample absorbances exceeds apredetermined absorbance, then disregarding all corrected sampleabsorbances.
 15. A method for measuring as claimed in claim 1, whereinthe comparison detects errors caused by one or more interferingcondition(s).
 16. A method for measuring as claimed in claim 15, whereinthe interfering condition(s) include air bubbles, finger prints, dirt ordefects in the cuvette.
 17. A method for measuring as claimed in claim2, wherein the analysis is performed on a diagnostic analyzer.
 18. Amethod for measuring as claimed in claim 1, wherein the light has awavelength in the range of 300 to 1100 nm.
 19. A method for measuringthe presence or concentration of an analyte in a sample by absorptionspectrophotometry, comprising: providing a cuvette having a sample withan analyte to be measured; providing a source of light and a detectorfor detecting the light; taking at least three measurements thatincludes: (i) directing at least three beams of the light to differentlocations on the cuvette; (ii) passing the at least three beams throughthe cuvette at their respective locations and through the sample to bemeasured; and (iii) measuring at least three respective sampleabsorbances of the transmitted beams with the detector; and comparingthe at least three sample absorbances to determine if: all the sampleabsorbances should be disregarded; one or more of the sample absorbancesshould be disregarded and the remaining absorbances retained; or all thesample absorbances should be averaged, wherein: if at least two sampleabsorbances are retained and an average retained absorbance is less thana first selected absorbance then the lowest absorbance is used indetermining the presence or concentration of the analyte; or if at leasttwo sample absorbances are retained and an average retained absorbanceis greater than or equal to a second selected absorbance then thehighest absorbance is used in determining the presence or concentrationof the analyte.
 20. A method for measuring as claimed in claim 19,wherein a single light source and a single detector are provided and thecuvette is moved relative to the light source and cuvette to produce theat least three beams of light.
 21. A method for measuring as claimed inclaim 19, wherein the first and second selected absorbances are both oneabsorbance unit.
 22. A method for measuring as claimed in claim 19,wherein the average retained absorbance is based on all sampleabsorbances.
 23. A method for measuring as claimed in claim 19, whereinprior to the step of directing at least three beams, the method furthercomprises: (i) directing at least three beams of the light at theirrespective different locations on the cuvette; (ii) passing the at leastthree beams through the cuvette alone or the cuvette and sample beforethe sample has reacted with reagents; and (iii) measuring at least threerespective blank absorbances of the transmitted beams with the detector;and selecting at least one blank absorbance; and subtracting at leastone blank absorbance from the at least three sample absorbances toresult in corrected sample absorbances.
 24. A method for measuring thepresence or concentration of an analyte in a sample by absorptionspectrophotometry, comprising: (A) providing a cuvette having a samplewith an analyte to be measured; (B) providing a source of light and adetector for detecting the light; (C) taking at least three measurementsthat includes: (i) directing at least three beams of the light todifferent locations a, b and c on the cuvette; (ii) passing the at leastthree beams through the cuvette at their respective locations a, b and cand through the sample to be measured; and (iii) measuring at leastthree respective sample absorbances Aa, Ab and Ac of the transmittedbeams with the detector; (D) determining the absolute value of thedifference between each pair of absorbances to arrive at |Aa−Ab|,|Ac−Ab| and |Ac−Aa|; (E) comparing an absolute value of the differencebetween each pair of absorbances with a predetermined limit; (F) if oneor more of each the absolute value of the difference is≧thepredetermined limit, then compare each absorbance to a predeterminedabsorbance: (i) if one or more absorbances are above the predeterminedabsorbance, then disregard all readings and proceed to step (K); or (ii)if all absorbances are below the predetermined absorbance, then (G)determine the smallest absolute value of the difference between eachpair of absorbances; (H) determine if the smallest absolute value of thedifference is<a predetermined fraction of the predetermined limit: (i)if the smallest absolute value of the difference is not less than thepredetermined fraction of the limit then disregard all readings andproceed to step (K); or (ii) if the smallest absolute value of thedifference is less than the predetermined fraction of the limit, then(I) determine which of the absolute value of the difference between eachpair of absorbances is the smallest absolute value of difference; (J)determine which absorbance in the smallest absolute value should beselected or if the results should be disregarded; and (K) eitherre-evaluating the analysis if the results should be disregarded in steps(F), (H) or (J), or calculating the presence concentration of theanalyte in the sample by using the selected absorbance.
 25. A methodaccording to claim 24, wherein prior to the step of directing at leastthree beams, the method further comprises: (i) directing at least threebeams of the light at their respective different locations a, b and c onthe cuvette; (ii) passing the at least three beams through the cuvettealone or the cuvette and sample before the sample has reacted withreagents; (iii) measuring at least three respective blank absorbances A1a, A1 b and A1 c of the transmitted beams with the detector; (iv)determining the sample absorbance Aa, Ab, and Ac by subtracting theblank absorbance A1 a, A1 b and A1 c from measured sample absorbance A2a, A2 b and A2 c respectively; wherein the step (J) of determining whichabsorbance in the smallest absolute value of the difference between eachpair of absorbances should be selected or if the results should bedisregarded comprises: (J1) if the smallest absolute value of thedifference between each pair of absorbances is |(A2 a−A1 a)−(A2 b−A1b)|, then if A1 c+A2 c is greater than each of A1 a+A2 a and A1 b+A2 b,compare A1 a+A2 a and A1 b+A2 b, if A1 a+A2 a<A1 b+A2 b then absorbanceAa is the selected absorbance, otherwise absorbance Ab is the selectedabsorbance, if A1 c+A2 c is≦to one of A1 a+A2 a and A1 b+A2 b, thendisregard all readings and proceed to step (K); (J2) if the smallestabsolute value of the difference between each pair of absorbances is|(A2 c−A1 c)−(A2 b-A2 b)|, then if A1 a+A2 a is greater than each of A1b+A2 b and A1 c+A2 c, compare A1 c+A2 c and A1 b+A2 b, if A1 c+A2 c<A1b+A2 b then absorbance Ac is the selected absorbance, otherwiseabsorbance Ab is the selected absorbance, if A1 a+A2 a is≦to one of A1b+A2 b and A1 c+A2 c, then disregard all readings and proceed to step(K); or (J3) if the smallest absolute value of the difference betweeneach pair of absorbances is |(A2 c−A1 c)−(A2 a−A1 a)|, then if A1 b+A2 bis greater than each of A1 a+A2 a and A1 c+A2 c, compare A1 c+A2 c andA1 a+A2 a, if A1 c+A2 c<A1 a+A2 a then absorbance Ac is the selectedabsorbance, otherwise absorbance Aa is the selected absorbance, if A1b+A2 b is≦to one of A1 a+A2 a and A1 c+A2 c, then disregard all readingsand proceed to step (K).
 26. A method according to claim 24, wherein thepredetermined fraction of the limit is 0.75 times the predeterminedlimit.
 27. A method according to claim 24, wherein locations a, b and c,correspond to left L, middle M and right R locations on the cuvette. 28.A method according to claim 24, wherein prior to the step of directingat least three beams, the method further comprises: (i) directing atleast three beams of the light at their respective different locationsa, b and c on the cuvette; (ii) passing the at least three beams throughthe cuvette alone or the cuvette and sample before the sample hasreacted with reagents; (iii) measuring at least three respective blankabsorbances A1 a, A1 b and A1 c of the transmitted beams with thedetector; (iv) determining the sample absorbance Aa, Ab, and Ac bysubtracting the blank absorbance A1 a, A1 b and A1 c from measuredsample absorbance A2 a, A2 b and A2 c, respectively.
 29. A methodaccording to claim 25, wherein the predetermined limit is equal tointercept+slope*(minimum of (A2 a−A1 a), (A2 b−A1 b) or (A2 c−A1 c)),wherein the intercept and slope are determined by the analyte beingmeasured.
 30. A method according to claim 25, wherein the predeterminedabsorbance is 1.0.
 31. A method according to claim 1 implemented by acomputer program interfacing with a computer.
 32. (canceled)